Aminoglutethimide inhibits steroidogenesis in the rat testis

Aminoglutethimide inhibits steroid formation in the adrenal and in the ovary but it is not established whether it has a similar effect in the testis. Adult male rats were injected with 10 mg or 15 mg amino-gluthethimide phosphate (AGP) per 100 g body weight twice daily for 3$\text{1}\text{2}$ days and killed 3 hrs or 5 hrs after the last injection. Treatment with either dose of AGP caused a precipitous decrease in plasma testosterone levels. Administration of the higher dose of AGP also caused a decrease in seminal vesicles weight and an increase in the concentration of esterified cholesterol in the testes. The results indicate that aminoglutethimide inhibits testicular steroidogenesis.     

Antiandrogenic and apoptotic effects of RU-486 on animal prostate

Mifepristone (RU-486) is a potent antagonist of steroid hormone receptors such as glucocoticoid and progesterone receptors. This compound also is a very strong inducer of the interaction between androgen receptors and corepressors NCoR and SMRT and therefore could be used as selective receptor modulator.

In this study we determined the relative binding affinity of RU-486 to androgen receptors (AR) obtained from rat prostate cytosol as well as the in vivo effect of different doses of RU-486 on the prostate weight of hamsters treated with dihydrotestosterone and/or RU-486. We determined also the prostate cell death (apoptosis) in hamster treated with, dihydrotestosterone (DHT) and/or RU-486.

The results of this study indicated that the relative binding affinity of RU-486 for AR is 4.3%. The data from the in vivo experiments also showed that RU-486 inhibited the prostate weight significantly in the highest doses thus indicating the antagonistic action of this compound on hamster prostate.

The immunohistochemistry analysis showed that after 1 month of castration, the hamster prostate was atrophic. Treatment with DHT produced epithelial cell activity (measured by the increase in the prostate weight) and very low rate of apoptosis. When DHT was administered together with RU-486 (10 mg/kg) no change was observed. On the other hand, DHT plus higher doses of RU-486 (40, 80 mg/kg) resulted in an increase of apoptosis in stromal and secretory epithelial cells. In addition to the increase of the prostate cell apoptosis produced by the treatment with high dose of RU-486, other factors could contribute to the decrease of the prostate weight observed. Another possibility could be a reduction in the ductal fluid due to poor epithelial cell secretory activity more than apoptosis itself. Furthermore, in this experiment, RU-486 could have inhibited the growth of the prostate gland produced by DHT in a greater extent than the induction of atrophy and cell death. This fact could depend on the doses used, due to the low affinity of this compound for the androgen receptors.     

Progesterone withdrawal and RU-486 treatment stimulate apoptosis in specific uterine decidual cells

Progesterone secretion is required for the growth and differentiation of endometrial stromal cells to form decidual cells. For many cells where a growth factor supports cell growth and proliferation, withdrawal of the growth factor initiates apoptosis. This study determined the time course and tissue location of apoptosis in deciduomal tissue after withdrawal of progesterone or injection of the antiprogestin, RU-486. Total DNA was isolated from decidual tissues at intervals after experimental treatments and separated electrophoretically. Internucleosomal DNA fragmentation characteristic of apoptosis was measured by quantitating levels of the 200 bp fragment. Apoptotic cells in tissue sections were detected by direct immunoperoxidase detection of digoxigenin-labeled DNA. Decidual apoptosis reached maximal levels at 12 h after withdrawal of progesterone or injection of RU-486. Increased concentrations of apoptotic cells were observed at the periphery of the growing deciduoma and in the antimesometrial deciduoma near the luminal epithelium after both treatments. These results suggest the withdrawal of progestin initiates apoptosis in cells at the early stages of decidualization.     

Verapamil inhibits proliferation but not steroidogenesis of regenerating rat adrenal cortex

The effect of verapamil, a calcium channel antagonist, on proliferation and steroidogenesis was investigated in regenerating rat adrenal cortex. Verapamil was given subcutaneously in two doses (1 and 5 mg/kg) to male Wistar rats subjected to adrenal enucleation combined with contralateral adrenalectomy. It was found that verapamil inhibited the mitotic activity of adrenocortical cells on the 4th and 8th day after surgery in a dose-dependent manner. However, no changes in corticosterone secretion were observed.     

Fear of black market means no RU-486 for Canada until US approves drug

Mifepristone, the "abortion pill" that is better known as RU-486, is no closer to arriving in Canada than it was 8 years ago. But that fact hasn''t slowed debate about the product.     

DNA binding properties of glucocorticosteroid receptors bound to the steroid antagonist RU-486.

RU-486 is an anti-fertility steroid which also has anti-glucocorticosteroid effects. RU-486 is shown to be a strong antagonist of the glucocorticosteroid-induced cytolytic response of the murine thymoma lines W7TB and T1M1b , and of the induction of mouse mammary tumor virus (MMTV) mRNA in T1M1b cells. The glucocorticosteroid receptor of W7 cells has high affinity for RU-486 (Kd = 3 X 10(-9) M) but the complex formed has low nuclear transfer capacity. Binding of RU-486, as compared with the glucocorticosteroid agonist triamcinolone acetonide, to mouse receptor results in a decreased affinity for DNA in general and a reduced specific recognition of a site in the promoter region of MMTV proviral DNA. The RU-486 complex formed with rat liver receptor exhibits the same behavior; in addition, it is shown that only a fraction of these complexes are activated by temperature and these form highly salt-sensitive interactions with DNA. These results indicate that the binding of RU-486 to glucocorticosteroid receptors mimics pharmacologically the properties of a class of receptor variants (nt-) which are non-functional and have reduced nuclear transfer and altered DNA binding capacity. These results substantiate the importance of DNA binding in receptor function.     

The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.     

Effect of the antiprogestin RU-486 on progesterone inhibition of occupied nuclear estrogen receptor in the uterus

The ability of the antiprogestin, RU-486, to reverse progesterone (P) antagonism of occupied nuclear E receptor retention was studied in the rat and hamster uterus. RU-486 was shown to effectively displace [3H]P binding from rat uterine cytosolic P receptor in in vitro competition assay. In contrast, no competition by RU-486 for [3H]P binding was observed for uterine cytosolic P receptor from the hamster uterus. In the presence of sustained serum levels (silastic implants) of P and estradiol (E), occupied nuclear E receptor was significantly inhibited in the rat uterus. At 6, 12 and 24h after RU-486 treatment (5 mg/animal, s.c.) uterine receptors for E and P were determined. No significant differences in cytosolic E and P receptors were observed between treated (E + P, + RU-486) and control (E + P alone) animals. However, by 6 h following RU-486 treatment, occupied nuclear E receptor retention increased significantly (0.30 +/- 0.05 vs 0.60 +/- 0.09, pmol/uterus) and reached a peak between 12 h (1.32 +/- 0.09) and 24 h (0.83 +/- 0.09). The increase in nuclear E receptor approached the level observed in animals with an E implant alone (1.55 +/- 0.15). Measurement of uterine fluid accumulation following RU-486 treatment showed an increase which paralleled that observed for occupied nuclear E receptor retention. A similar in vivo experiment in the hamster showed no reversal of P inhibition of occupied nuclear E receptor. These results show that: 1. RU-486 is an effective competitor for rat uterine P receptor but not hamster P receptor; 2. RU-486 can rapidly reverse P inhibition of uterine occupied nuclear E receptor in the presence of sustained serum levels of E and P; 3. The recovery of occupied nuclear E receptor is coincident with a resumption of E action (uterine fluid accumulation). The studies also provide a novel means by which antiprogestin activity can be assessed in vivo in the presence of sustained E and P serum levels, e.g. the reversal of P inhibition of uterine nuclear E receptor retention.     

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.     

Co-administration of etomoxir and RU-486 mitigates insulin resistance in hepatic and muscular tissues of STZ-induced diabetic rats.

Insulin resistance is a condition of central importance in a cluster of clinical disorders including diabetes mellitus, hypertension, dyslipidemia, central obesity and coronary heart disease. Despite its association with numerous health problems, the mechanism responsible for the development of this phenomenon remains to be established. A novel theory has proposed that insulin resistance in diabetes stems, at least in part, from enhanced free fatty acid (FFA) oxidation and/or excessive production of glucocorticoids (GCs). Several key predictions of this premise were subjected to experimental testing using streptozotocin (STZ)-treated rats as a model for insulin-dependent diabetes mellitus and euglycemic-hyperinsulinemic clamp technique for the in vivo measurement of insulin actions. Euglycemic clamp studies with an insulin infusion index of 5 mU/kg/min were used to measure endogenous glucose production (EGP), glucose infusion rate (GIR), glucose disposal rate (GDR) and skeletal muscle glucose utilization index (GUI). Post-absorptive basal EGP and plasma levels of glucose and free fatty acids (FFA) were elevated in the STZ diabetic rats compared to their corresponding control values. In contrast, hypoinsulinemia was evident in these animals. Steady-state GIR and GDR during euglycemic-hyperinsulinemic clamp were markedly decreased in the STZ diabetic rats. Similarly, insulin-mediated suppression of EGP and plasma FFA concentration was also impaired in these animals. GUI, a measure of 2-deoxyglucose (2-DG) uptake, was increased in response to insulin in the order of white gastrocnemus (WG), red gastrocnemus (RG), extensor digitorum longus and soleus muscles. This parallels the percentage of red fibers in these muscles. Diabetes interferes with insulin's ability to increase 2-DG uptake in all of the above muscles with the exception of WG. Nullification of the associated hyperlipidemic and hypercortisolemic states of diabetes with etomoxir (hyperlipidemic) and the glucocorticoid receptor blocker RU-486 (hypercortisolemic) ameliorated the diabetes-related impairment of the in vivo insulin action. Overall these results together with those garnered from the literature support the notion that hypercortisolemia and the enhancement of FFA oxidation are involved, at least in part, in the development of hepatic and skeletal muscle insulin resistance in poorly controlled type I diabetes.     

LH-RH antagonist inhibits gonadal steroid secretion in vitro

This investigation was aimed at studying the direct action of LH-RH derivatives on gonadal function. Modulation by LH-RH antagonist (Ac-[D-beta-Nal1, D-p-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10]-LH-RH) and agonist of LH-RH (D-Ser(TBU)6, AzaGly10-LH-RH) and native LH-RH of HCG-stimulated steroidogenesis in testicular Leydig cells and luteal cells was studied in vitro. The LH-RH antagonist (3.2 X 10(-8) M) was found to change ED50 of HCG from 2 X 10(-11) M to 5.5 X 10(-11) M in the Leydig cell culture system. In addition, the antagonist was noted to override the stimulatory action of native LH-RH in Leydig cells. Furthermore, the agonist was found to augment HCG-provoked testosterone secretion. Similarly, the LH-RH antagonist at 10(-9) M blunted HCG-stimulated progesterone secretion in the luteal cell culture system and increased the ED50 of HCG from 8.7 X 10(-13) to 7.7 X 10(-12) M. In contrast to the Leydig cell culture system native LH-RH (10(-8) M) and the agonist (10(-8) M) increased the ED50 of HCG in luteal cells from 8.7 X 10(-13) M to 3 X 10(-12) M and 2.3 X 10(-12) M, respectively. Present data combine to suggest that LH-RH antagonists act at least partially at the gonadal level and may be clinically useful to inhibit Leydig cell and ovarian function.     

Acute starvation affects rat adrenal steroidogenesis

To determine how starvation affects adrenal steroidogenesis we measured the activities of 3 adrenal enzymes involved in corticosterone biosynthesis in a group of adult female rats. The animals were either starved for 7 days or fed ad libitum for the same period. Relative adrenal weight and plasma corticosterone levels were increased in the experimental group of animals compared to the control group (40 +/- 2 vs 27 +/- 1 mg/100 g body weight, P less than 0.001, and 45 +/- 4 vs 30 +/- 5 ng/dl, P less than 0.05 respectively). There were no differences in plasma ACTH levels between the groups (34 +/- 5 vs 26 +/- 4 pg/ml). 11-Hydroxylase activity was increased in the starved group of animals (18 +/- 3 vs 8 +/- 2 nmol/mg protein/min, P less than 0.01). 3 beta-Hydroxysteroid dehydrogenase and 21-hydroxylase activities were not different between the groups (19 +/- 2 vs 16 +/- 1 nmol/mg protein/min, and 100 +/- 10 vs 110 +/- 10 pmol/mg protein/min respectively). These results suggest that acute starvation in rats produces an increase in adrenal 11-hydroxylase activity.     

Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles.

The effect of cantharidin, a natural toxicant of blister beetles and a strong inhibitor of protein phosphatases types 1 and 2A, on luteinizing hormone (LH)-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450scc) enzyme. Treatment of preovulatory follicles dissected from ovaries of immature rats primed with pregnant mares' serum gonadotropin (10 IU) with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 100 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of follicles with increasing concentrations (10 - 1000 ng/ml) of cantharidin suppresssed LH (100 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450scc protein and the conversion of 22R-hydroxycholesterol to progesterone were not affected by cantharidin. This indicates that cantharidin did not interfere with the activity of P450scc. Cantharidin also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (10(-5) M) or a cAMP analog 8-Br-cAMP (0.5 mM). These results demonstrate that cantharidin inhibits the LH-induced StAR protein levels, and, thus, suggest that phosphoprotein phosphatase activity is required for the cAMP-protein kinase A-stimulated steroidogenic activity of the preovulatory follicle.     

Alterations in gonadal steroidogenesis in individuals expressing a common genetic variant of luteinizing hormone

Some pathologies of the pituitary-gonadal function have recently been found to be due to mutations of the gonadotropin or gonadotropin receptor genes. Although these conditions are extremely rare, they are very informative, by elucidating some less well characterized facets of normal gonadotropin function and the molecular pathogenesis of disturbances in sexual differentiation and fertility. In contrast, there is a common polymorphism in the Luteinizing Hormone (LH) β-subunit gene, where two point mutations cause two alterations in the amino acid sequence (Trp⁸→Arg and Ile¹⁵→Thr) and introduce an extra glycosylation signal to Asn¹³. The carriers of this variant gene are largely healthy, but certain mild differences in their gonadal function have been found, as reflected by alterations in gonadal steroidogenesis, pubertal development and predisposition to diseases such as infertility, polycystic ovarian syndrome, and breast and prostatic cancer. The purpose of this chapter is to review the current knowledge of the occurrence, special functional features and clinical correlates of this LH variant.     

Transforming growth factor-beta inhibits Leydig cell steroidogenesis in primary culture

The effects of transforming growth factor (TGF) on Leydig cell steroidogenesis in primary culture were investigated. Basal testosterone levels were 3.7 +/- 0.54 ng/ml (mean +/- SE, N = 7). In the presence of hCG (10 ng/ml), testosterone levels increased to 22.77 +/- 3.05 ng/ml. TGF-beta caused a dose dependent inhibition of hCG-stimulated testosterone formation but without effects on basal levels. TGF-beta also inhibited 8-bromo cyclic AMP-induced testosterone formation and hCG-stimulated cyclic AMP formation. In contrast, TGF-alpha had no effect on either basal or hCG-stimulated testosterone formation and did not modify the inhibitory effect of TGF-beta. Present study indicates that TGF-beta can modulate Leydig cell steroidogenesis.     

Disrupting mitochondrial function with surfactants inhibits MA-10 Leydig cell steroidogenesis

It is well established that surfactants can elicit cytotoxic effects at threshold concentrations by changing the permeability and solubilizing components of cell membranes. The purpose of this study was to characterize the relationship between perturbation of the mitochondrial membrane resulting from treatment with representative cationic, nonionic, and anionic surfactants and the extent to which this perturbation affects steroid formation and StAR protein expression and activity in MA-10 Leydig cells. The StAR protein is synthesized as an active 37 kDa extramitochondrial form, which is processed into a 30 kDa intramitochondrial form after cholesterol transfer and mitochondrial import and processing. It has been shown in several in vitro studies that the mitochondrial electrochemical gradient is required for the StAR protein to transfer cholesterol to the inner mitochondrial membrane. Each substance that was tested produced a concentration-dependent decrease in steroid formation in hCG-stimulated MA-10 cells. Decreases in progesterone production were accompanied by loss of mitochondrial membrane potential and by a decrease in the levels of the 30 kDa form of the StAR protein. However, levels of the 37 kDa form of the StAR protein did not decrease, indicating no effect on StAR protein expression. These results demonstrate how perturbation of the mitochondrial membrane by surfactants inhibits import, processing, and cholesterol transfer activity and underscore the importance of including sensitive assays that evaluate mitochondrial function when screening for potential effects on steroidogenesis with in vitro test systems.     

Cyclic AMP and steroidogenesis in superovulated rat ovaries

During the gonadotropin stimulated differentiation of ovarian follicles into corpora lutea, the concentrations of total cyclic AMP and protein bound cyclic AMP increased only marginally. In contrast, there was a 10 fold increase in progesterone production. After acute stimulation of luteinized ovaries with choriogonadotropin total cyclic AMP levels increase more than 6 times while bound cyclic AMP and plasma progesterone rose by 80 and 60% respectively. Our results question the role of cyclic AMP in the basal production of progesterone by the ovary, and suggest a relationship between bound cyclic AMP and progesterone synthesis only exists after acute gonadotropin stimulation.